In this study, we thoroughly explore the clinical and biological features of immunotherapy-induced vitiligo-like depigmentation (VLD) in patients with stage III-IV melanoma receiving immunotherapy. Key findings include a distinct immune signature with an upregulation of ITGA in the VLD group, an upregulation of EDAR and downregulation of LAG3 in VLD-responders group, a longer time to VLD onset in patients pre-treated with targeted therapy, the distribution of VLD lesions primarily in sun-exposed areas as opposed to mechanically stressed areas in non-immunotherapy-related vitiligo, the predominance of a symmetrical, small-freckle lesional pattern and a prolonged progression-free survival (PFS) and overall survival (OS) in VLD patients without LDH elevation as opposed to those with LDH elevation.Vitiligo-like depigmentation (VLD) is an immune-related adverse event (irAE) of checkpoint-inhibitor (CPI) treatment, which has previously been associated with a favourable outcome. The aim of this study was to explore clinical, biological and prognostic features of melanoma patients with VLD under CPI-treatment and to explore whether they exhibit a characteristic immune response profile in peripheral blood. Melanoma patients developing VLD under CPI were included in a prospective observational single-center cohort study. We collected and analysed clinical parameters, photographs and serum from 28 VLD patients. They received pembrolizumab (36%), nivolumab (11%), ipilimumab/nivolumab (32%) or clinical trial medications (21%). We performed a high-throughput proteomics assay (Olink), in which we identified a distinct proteomic signature in VLD patients in comparison to non-VLD CPI patients. Our clinical assessments revealed that VLD lesions had a predominantly symmetrical distribution pattern, with mostly smaller “freckle-like” macules and a preferential distribution in UV-exposed areas. Patients with previous targeted therapy showed a significantly longer time lapse between CPI initiation and VLD onset compared to non-pre-treated patients (12.5 vs. 6.25 months). Therapy responders exhibited a distinct proteomic profile when compared with non-responders in VLD such as upregulation of EDAR and downregulation of LAG3. ITGA11 was elevated in the VLD-group when compared to non-VLD-CPI-treated melanoma patients. Our findings demonstrate that on a proteomic level, VLD is characterized by a distinct immune signature when compared to CPI-treated patients without VLD and that therapy responsiveness is reflected by a characteristic immune profile. The pathomechanisms underlying these findings and how they could relate to the antitumoral response in melanoma remain to be elucidated. Checkpoint inhibitors (CPI) and targeted therapies have revolutionized the treatment for metastatic melanoma over the past decade ,2,3,4. TCTLA-4 and PD1 are both regulators of the T-cell response . CTLA-4 VLD occurs in 4.7–37.5% of the melanoma patients undergoing anti-PD1 treatment and in 2.9–4.9% of the melanoma patients treated with CTLA-4-antibodies ,14,15,16There have been attempts to characterize VLD on a cytokine level ,20. HoweMany questions remain to be addressed about VLD in melanoma. The aim of this prospective study was to explore the proteomic immune signature of VLD, to characterize its clinical presentation and to investigate the potential association with the clinical response under CPI. This monocentric, prospective observational cohort study was performed at the Department of Dermatology of the University Hospital Zurich, Switzerland. Adult melanoma patients (≥18 years) treated with CPI who developed VLD were included between 05/2018 and 08/2020. Our study population consisted of four different treatment cohorts receiving the following immunotherapies: a combination of ipilimumab and nivolumab, anti-PD-1 monotherapy with pembrolizumab or nivolumab and clinical trials. All patients received immunotherapies according to standard dose and schedule. Prerequisites for inclusion were advanced melanoma and CPI-associated VLD. Exclusion criteria for the subanalysis regarding responsiveness included adjuvant immunotherapy. One additional patient was eliminated from the analysis due to unclear data.At two to three-week interval visits, patients were screened for VLD manifestations by a dermatologist. VLD was defined as the appearance of depigmented skin lesions and categorized as symmetrical vs. asymmetrical, small macules (≤1 cm) vs. large patches (>1 cm), with a universal, polymorphous, or confluent distribution. Macules, as well as patches, are flat, non-elevated lesions which are only distinguished by their size. The clinical course of VLD, including the body surface area (BSA) measurement, was systematically recorded by photography of affected areas. In addition, Wood’s light examination was used to confirm the diagnosis. The photographs were analysed as to the extent of VLD (affected BSA) and localization  by two independent blinded investigators. In addition to VLD, all irAEs were documented according to the current standards (Common Terminology Criteria for Adverse Events CTCAE v4.03). Regular stagings with PET-CT were performed every three months using RECIST criteria (version 1.1)  accordinThe study was conducted according to the Helsinki Declaration and was approved by the local ethics committee (EK2019-01825). Data protection, according to the EU and Swiss standards, was guaranteed and enforced for all study patients.For serological protein analysis, OLINK assays were performed using two panels: Inflammation and Immune Response . The proximity extension assay (PEA) was performed according to manufacturer’s instructions. Briefly, protein-specific antibodies tagged with DNA sequences were used, followed by a PCR where matched DNA reporter pairs served as the template. In the last step, a Biomark HD system  device was used for microfluidic qPCR and therefore signal quantification. Data were analysed using R 4.1.2  and the package OlinkAnalyze 3.1.0  and OlinkR1.0.1  as well as for visualisation ComplexHeatmap 2.10.0  and EnhaIn all patients, the diagnosis of VLD was histopathologically confirmed by a board-certified dermatopathologist. Loss of melanin pigmentation and melanocytes were evaluated in nine patients using Masson Fontana stain and immunohistochemical stainings . t-test for independent groups. In case of confirmed variance homogeneity and to determine the significance, a 2-sided t-test was performed. The significance was calculated with a confidence interval of 95%. For the pattern analysis, an Anova test was applied. For survival analyses, a Schoenfeld’s test was conducted to check the proportional hazards assumption, a Kaplan-Meier estimator used to plot survival curve and the log-rank test applied to assess if the curves were significantly different. Descriptive statistics and visualizations were elaborated in RStudio. For further statistical evaluations, such as the examination of variance homogeneity and significance calculations, SPSS was used. For the variance homogeneity examination, Levene’s test was applied, a t-test was used, whilst multiple groups were analysed by applying an ANOVA F-test. The Benjamini–Hochberg method (“fdr”) was used for the multiple testing correction. Heatmaps and volcano plots were created using the limma function of the OlinkR package as well as the EnhancedVolcano and ComplexHeatmap packages. Analyses of the Olink protein data were processed applying the “OlinkAnalyze” R package provided by Olink . For the comparison of two groups, a A total of 28 VLD patients were included with a mean observation period of 26.8 months ± 19.46 months. The mean age of patients at VLD diagnosis was 66.04 ± 13.50 years. 68% of the VLD patients were male, and the male-to-female ratio was approximately 2:1. At the time of diagnosis, male patients were slightly older (66.84 ± 13.01 years) in comparison to female patients (64.33 ± 15.15 years). All patients suffered from stage IIIB-IV melanoma at the time of VLD diagnosis  (p = 0.19) . We systemically assessed lesions with regard to their morphology and distribution. We observed a significantly higher percentage of patients with a symmetrical rather than with an asymmetrical distribution of VLD lesions (≤ 0.001) . Slightl = 0.19) . In all  = 0.19) E. All pa = 0.19) D. Three  = 0.19) F.Histopathological assessment of nine VLD patients demonstrated a complete loss of melanin pigmentation (demonstrated with Masson-Fontana stain) in 9/9 cases. Melanocytes  were completely absent in 5/9 (55.5%) and significantly reduced in number in 4/9 (44.4%) specimens. Next, we aimed to investigate the responsiveness of VLD patients to immunotherapy. Our patients had received a combination of ipilimumab and nivolumab , anti-PD-1 monotherapy with pembrolizumab  or nivolumab (3/11%), other immunotherapy-based clinical trial medications  or anti-CTLA-4 monotherapy prior to VLD onset. Excluding the adjuvant patients and one patient with unclear data, the complete remission rate was 24% (n = 6), partial remission rate was 28% (n = 7), stable-disease rate was 8% (n = 2) and progressive disease rate was 40% (n = 10) at VLD-onset. Three years following VLD onset, the probability for progression-free survival was 60.4% (95% CI: 43–84%) A and thep ≤ 0.001). The median time until VLD onset in the non-pre-treated group was 6.25 months (range 1–13 months) and the median average time in the pre-treated group was 12.5 months (range 9.5–16 months)  months; the median time lapse was 6.5 months. We compared patients who had received a targeted therapy beforehand (n = 4) to patients who had not (n = 20). Four patients were excluded from this analysis due to missing data. Patients who had previously received targeted therapy developed VLD significantly later (6.5–7.2 months) than patients with no prior treatment ( months) A.We were interested in whether we might identify particular clinical or molecular parameters associated with a better treatment response among our VLD patients. Regarding the clinical VLD pattern , we did not find any differences between them in terms of PFS or OS (p = 0.81)  had a mean time of 6.92 (±3.16) months and median time of 6.5 months until VLD development, vs. a mean time of 7.58 (±4.28) months and median time of 7.5 months until VLD onset in the non-responders group (n = 12). The observed differences were not significantly different ( = 0.81) B.p = 0.49). A normal serum lactate dehydrogenase (LDH) was associated with improved outcome   .In this study, we explored the systemic immune signatures and characterized clinical patterns of VLD and evaluated whether they are associated with different treatment outcomes in melanoma patients receiving CPI .Our proteomic analysis revealed a distinct immune signature of VLD in comparison to CPI recipients without VLD, both with/without other irAEs. Of note, several proinflammatory proteins, such as CXCL5, CCL11 and TNFSF14, were significantly reduced in VLD. There were no clear differences investigating nVLD with and without other irAEs; however, the sample size was limited regarding a direct comparison. Based on these data, one may hypothesize that a specific constellation of cytokines may induce or indicate VLD. Of note, ITGA11 especially was increased in VLD patients. ITGA11 belongs to the group of integrins which are transmembrane proteins that mediate cell adhesion to the extracellular matrix . They plClinically, VLD preferentially develops in sun-exposed areas. The sun-exposed area-distribution seems to be a VLD-specific phenomenon which is not commonly observed in non-immunotherapy-associated vitiligo ,20,26. OHistopathological evaluation revealed findings compatible with classical spontaneously occurring vitiligo ,33, i.e.The clinical pattern analysis of VLD revealed a predominance of symmetrically distributed macular depigmented lesions, whereas patches and an asymmetrical pattern were less prevalent. Based on our findings, we hypothesized that this particular VLD pattern may be associated with a distinct underlying T-cell response and thus a different clinical outcome. This theory was not confirmed by our analysis of outcome in terms of OS and PFS, which was not different in VLD with vs. without a macular pattern. We did, however, find that biochemical factors (a low baseline LDH) correlated with a significantly better outcome (PFS and OS). Our findings confirm the association of an LDH above the upper normal limit with an inferior outcome, which has previously been observed in non-VLD ICP cohorts ,35. The Interestingly, responders vs. non-responders within the VLD group showed much more heterogeneity in their proteomic immune profiles than those without VLD. Responders showed several increased proteins, such as EDAR and PLXNA4, whereas LAG3 and other proteins were reduced. LAG3 is an inhibitory molecule that is targeted by novel treatments (relatlimab) . The dowWe compared our data to the TCGA database and no relevant information regarding the according genes to the significant up- and downregulated proteins was found. While this study includes a large VLD cohort in a single center, caution is warranted when interpreting our results due to several limitations. For the targeted high-throughput proteomics (Olink) experiments, the sample size of the control group was quite small (n = 7), since in the current project we focused on the characterization and stratification within the VLD group itself. Our presented findings need to be confirmed in larger cohorts, which should include larger control groups without VLD, but only with other irAEs and without any irAEs. The heterogeneity of our VLD patient population could be an additional source of bias and underlines the need for further sub-analyses according to different tumor stages, CPI combinations and pre-treatments. Future studies should seek to investigate the different pathomechanism underlying the development of VLD compared to classical vitiligo to better characterize the T-cell-mediated mechanism in both conditions.Our results highlight the distinct proteomic immune signatures, such as an upregulation of EDAR and downregulation of LAG3, in responders among VLD melanoma patients in comparison to non-responders. The clinical characterization of VLD in melanoma patients has revealed a predominance of a symmetrical, small macular pattern of depigmentation in UV-exposed areas without a specific pattern showing an advantage in tumor response or survival. The significantly longer time to VLD onset in the kinase-inhibitor pre-treated group is an additional interesting new finding which gives us clues as to the immunological factors contributing to VLD. Futures studies with larger sample sizes should investigate how our findings relate to the antitumoral immune response and what we could extrapolate from this to the clinics.


These characteristics are decisive features for application as a matrix in fiber-reinforced polymer composites produced via prepreg technology in which low viscosity and sufficient latency, meaning low reactivity of the one-component system, are necessary. The homogeneous mixture of amino acid and epoxy resin was prepared via three-roll milling. Two formulations, Argopox-1 with 1 The need for sustainable practices in the processing chain of fiber-reinforced thermosets has led to the development of bio-based epoxy resins and curing agents. As a contribution to sustainable composites, this study focuses on the glass transition temperature ( Therefore, substituting petroleum-based compounds with bio-based alternatives results in lower CO2 emissions and thus contributes to climate protection [Glass and carbon fiber reinforced epoxy matrix composites play an important role in industries such as sports, automotive, wind energy, and aerospace due to their high weight-specific strength and modulus . Here, potection .2) and carboxylate (-COOH) functional groups, as well as a side chain (-R) that is distinct for each amino acid  anl-tryptophan [l-tryptophan cured DGEBA ranges from 66 °C to 101 °C if ureas were used as accelerator [l-tryptophan and DGEBA was accelerated by imidazoles which resulted in glass transition temperatures of 84 °C to 104 °C [l-tryptophan. However, none of the previous investigations, except Shibata et al. [R of active hydrogen atoms from l-tryptophan to the epoxy groups of the resin.Previous investigations on the potential of amino acids as bio-based curing agents focused mainly on the reaction kinetics and glass transition temperature yptophan ,12,13,14o 104 °C ,13,15. Ta et al. , used a l-tryptophan looks like a promising curing agent due to its aromatic structure that poses steric hindrance between cross-links, thus preventing rearrangement of network segments between cross-links which in turn results in a high l-tryptophan is only four  was used as the resin. l-arginine with a purity of 98.9% was bought from Buxtrade GmbH . The reaction between amino acid and epoxy resin was accelerated by the substituted urea DYHARD® UR500 by Alzchem Group AG .D.E.R. 331 with an epoxide equivalent weight of 187 g moll-arginine has seven active hydrogen atoms. Therefore, the amine equivalent weight of l-arginine is approximately 24.9 g mol−1. A homogeneous mixture (stoichiometric ratio R = 1) of D.E.R. 331 and l-arginine was prepared using a dual asymmetric centrifuge Speed mixer by Hauschild Engineering  at 3000 min−1 for 120 s.Similar to the investigation by Shibata et al. , it was l-arginine is processed using a three-roll mill type EXAKT 120 EH-450 by EXAKT Advanced Technologies GmbH . This method has already been applied for dispersing different fillers in epoxy resin [−1. The numbers of revolutions of the feed roll and middle roll were set automatically by the three-roll mill. Here, the ratios of the number of revolutions of feed roll to middle roll to apron roll are 1:3:9. The small gap combined with the high number of revolutions leads to shear rates in the gap in the order of magnitude of 105 s−1. The stress exerted by the fluid on the particles results in a break-up of particles. The mixture was run through the three-roll mill five times to ensure sufficiently small particle sizes and a narrow particle size distribution.To decrease the particle size of the amino acid, the mixture of DGEBA and xy resin ,25 and pxy resin . The tem−1 for 120 s. Afterwards, the formulations were kept in an vacuum oven at 45 °C and 20 mbar for 1 h to ensure the elimination of entrapped air.Afterwards, two different formulations, one with 1 ared see . For theOne part of each formulation was stored at room temperature  plate-plate rheometer. The plate diameter was 25 mm and the gap between plates was set to 1 mm. A constant heating rate of 3 K minThe cured thermoset was characterized by a Zeiss Gemini 1530 Scanning Electron Microscope by Carl Zeiss AG . The acceleration voltage used was 3 kV and the surfaces were platinum-sputtered with a thickness of about 5 nm.l-arginine without any accelerator  as well as with 1 ® UR500. With the addition of the accelerator, the exothermic peak of the heat flow becomes smaller but stays at around 208 °C  [l-arginine.The vely see b. The dil-tryptophan [l-arginine which contains three carbon atoms between its The yptophan ,13,14,15d 140 °C ,28. The ® UR500 as result of aging at room temperature after 91 d [l-arginine-cured epoxy resin is less latent, meaning more reactive, than the petroleum-based material system. For the epoxy cured with dicyandiamide, the increase in l-arginine, it is likely that the same mechanisms are at work, meaning that the shift in ® UR500.elerator . The resAs already mentioned above, the change in Contrary to that, the viscosity function of Argopox-1 shows no systematic trends regarding its change in dependence of storage time during storage at 8 °C see . Here, tl-arginine in the epoxy resin has to be considered. The processing via three-roll milling leads to a decreased average particle size and a narrow particle size distribution. l-arginine particles with size between approximately 50 to 100 nm can be observed (see To find a reasonable explanation for the observed double peak in the viscosity function of Argopox-1, the morphology of rved see d. This ml-arginine in DGEBA. As the formation of the double peak is different for Argopox-1 and Argopox-2.5 (see ® UR500. However, the exact mechanism behind the emergence of the double peak in viscosity is beyond the scope of this investigation and might be focused upon in the future.In general, the viscosity of a suspension depends, among other things, on the viscosity of the matrix, the particle volume fraction as well as particle aspect ratio. Another influencing factor might be the temperature-dependent solubility of -2.5 see , it is rSimilarly to the results of Argopox-1, the viscosity of Argopox-2.5 shows a peak in its fresh state at around 132 °C see . HoweverInterestingly, storage at −18 °C has a more systematic influence on the viscosity of Argopox-2.5 than on that of Argopox-1 see . Here, tl-arginine cured DGEBA in the presence of a urea-based accelerator. A solvent-free processing method for the mixture of epoxy resin and amino acid via three-roll milling was presented and its suitability as a preparation method was confirmed via DSC. The l-arginine is considerably lower than that of dicyandiamide due to the aliphatic side chain of l-arginine. The DSC and SAOS measurements show that the This study focused on the glass transition temperature, viscosity and latency of


Background and Objectives: The aim of this paper is to evaluate the impact of humoral substance mid-regional pro-adrenomedullin (MR-proADM) on the two-year survival of patients with chronic heart failure and relate it to the dosage of furosemide. Materials and Methods: The data is taken from the stable systolic heart failure (EF < 50%) FAR NHL registry (FARmacology and NeuroHumoraL activation). The primary endpoint at two-year follow-up was death, heart transplantation, or LVAD implantation. Results: A total of 1088 patients were enrolled in the FAR NHL registry; MR-proADM levels were available for 569 of them. The mean age was 65 years, and 81% were male. The aetiology of HF was ischemic heart disease in 53% and dilated cardiomyopathy in 41% of patients. The mean EF was 31 ± 9%. Statistically significant differences (p < 0.001) were obtained in several parameters: patients with higher MR-proADM levels were older, rated higher in NYHA class, suffered more often from lower limb oedema, and had more comorbidities such as hypertension, atrial fibrillation, diabetes, and renal impairment. MR-proADM level was related to furosemide dose. Patients taking higher doses of diuretics had higher MR-proADM levels. The mean MR-proADM level without furosemide (n = 122) was 0.62 (±0.55) nmol/L, with low dose (n = 113) 1–39 mg/day was 0.67 (±0.30) nmol/L, with mid dose (n = 202) 40–79 mg/day was 0.72 (±0.34) nmol/L, with high dose (n = 58) 80–119 mg/day was 0.85 (±0.40) nmol/L, and with maximum dose (n = 74) ≥120 mg/day was 1.07 (±0.76) nmol/L, p < 0.001. Patients with higher MR-proADM levels were more likely to achieve the primary endpoint at a two-year follow-up (p < 0.001) according to multivariant analysis. Conclusions: Elevated plasma MR-proADM levels in patients with chronic heart failure are associated with an increased risk of death and hospitalization. Higher MR-proADM levels in combination with increased use of loop diuretics reflect residual congestion and are associated with a higher risk of severe disease progression. Chronic heart failure (HF) is a clinical syndrome with a serious prognosis and increasing incidence. It is characterized by typical symptoms  that may be accompanied by signs  .Plasma concentrations of natriuretic peptides are recommended as initial diagnostic tests in patients with symptoms suggestive of HF in order to rule out the diagnosis . They caAdrenomedullin (ADM) is a member of the calcitonin gene-related peptide (CGRP) superfamily . It is sADM circulates in the plasma in very low concentrations in healthy humans; however, it rapidly increases in patients with heart failure . NeverthThese data come from the FAR NHL registry (FARmacology and NeuroHumoraL activation), which is a database of patients with stable systolic heart failure (EF < 50%). Medical history data, and physical examination and biochemistry results including NT-proBNP were collected from seven departments with specialized heart failure care in three university hospitals in the Czech Republic between November 2014 and November 2015.The primary endpoint, i.e., the two-year prognosis in terms of all-cause mortality, heart transplantation, and/or left ventricular assist device (LVAD) implantation, was evaluated up to November 2017. Patients were followed up prospectively at outpatient departments and mortality rates were verified in the National Death Certificate System. Monitored data on all patients were gathered at the end of the two-year follow-up. Eligible patients had to be treated for heart failure with non-preserved ejection fraction (EF < 50%) and to be stable for at least one month with an optimal pharmacotherapy treatment, as recommended in ESC guidelines for chronic heart failure. Exclusion criteria were not signing the informed consent, signs and symptoms of acute decompensation of heart failure, and conditions other than heart failure that would certainly limit the mid-term prognosis of patients . Patients with an orthotopic heart transplant (OHT) or with a left ventricle assistance device (LVAD) implantation in the baseline were not included.The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of Brno University Hospital, Czech Republic .In the whole FAR NHL registry, a total amount of 1088 patients were included. The level of MR-proADM was available in 569 of them. The mean age was 65 years, 80.7% were male, the aetiology of chronic HF was ischemic heart disease in 53.3%, and dilated cardiomyopathy in 40.5% and 6.2% were classified as other. Mean EF was 31 + 9%. The most common comorbidities were hypertension (64.1%) and diabetes mellitus (40.2%). Beta-blockers were administered to 93.1% of patients, drugs blocking the renin-angiotensin system (ACE inhibitors and/or ARBs) were administered to 88.4% of patients, and diuretics were administered to 88.0% of patients. The most prescribed diuretic was furosemide, which was given to a total of 78.6% of patients alone or in combination, followed by spironolactone or eplerenone, which was prescribed to 64.1% of patients, and hydrochlorothiazide 12.8%. Patients were classified as NYHA I at 13.7%, NYHA II at 66.3%, and NYHA III-IV at 20.0%. The baseline characteristic of the cohort is displayed in Standard descriptive statistics were applied in the analysis; continuous variables were described by mean ± SD or in the case of laboratory results by median and interquartile range, whereas categorical variables were characterized by absolute and relative frequencies. Statistical significance of differences among groups of patients was analysed using the Mann–Whitney test or the Kruskal–Wallis test for continuous variables and Fisher’s exact test for categorical variables.p < 0.05) were entered into a multivariable logistic regression model with a backward stepwise algorithm for the selection of independent predictors of the primary endpoint. The analysis was performed using SPSS version 26.0 .The predictive power of MR-proADM for the combined endpoint was analysed using ROC analysis and described by AUC, its confidence interval, and statistical significance. The optimal cut-off was selected by maximization of the Youden index. Time to combined endpoint was visualized using the Kaplan–Meier methodology and the computation of the proportion of surviving patients and its 95% confidence interval in a given time point was also based on Kaplan–Meier estimates. Statistical significance of differences in time to the event among groups of patients was evaluated using the log-rank test. Parameters were assessed using univariable logistic regression models. Significant factors  technology.The plasma samples were collected, directly frozen, and stored at −80 °C. Biomarker of cardiovascular load—MR-proADM—was detected using the homogeneous sandwich fluor immunoassay on the automated KRYPTOR . The B·R·A·H·M·S KRYPTORThe detection limit of the kit B·R·A·H·M·S MR-proADM KRYPTOR  was 0.05 nmol/L, and the direct measurement range was 0.05–10 nmol/L, enhanced with the use of automatic dilution up to 100 nmol/L. The functional assay sensitivity (inter-assay precision of 20% CV) has been assessed as being 0.25 nmol/L. In the concentration range of >2–6 nmol/L was the intra-assay of <2% CV, and the inter-assay of ≤10% CV.p < 0.001). The statistically significant difference (p < 0.001) was obtained in these parameters according to the level of MR-proADM: patients with a higher level of MR-proADM were older, evaluated by higher NYHA class, suffer more often from swollen lower limbs, and had more comorbidities such as hypertension, atrial fibrillation, diabetes mellitus, and renal dysfunction. The complete results are displayed in Patients without a primary endpoint were assigned to group A (479 pts), and those with the primary endpoint to group B (86 pts). A total of 10 patients were LVAD implanted, 16 patients underwent heart transplantation and 66 patients died. Some of the patients from group B reached more than one endpoint in general. There were statistically significant differences between the groups in the levels of MR-proADM: group A median 0.62 nmol/L  vs. group B 0.88 nmol/L  (p < 0.001). Multivariable analysis has revealed that the dose of furosemide and MR-proADM are independent predictors of the primary endpoint. , ndpoint. . Althougndpoint. . ROC curp < 0.001 .The distribution of MR-proADM levels in patients from our database during the two-year occurrence of the combined endpoint is displayed in n = 122) was 0.62 (±0.55) nmol/L, compared to patients with the maximum dose furosemide (n = 74) ≥ 120 mg/day where the mean level of MR-proADM was 1.07 (±0.76) nmol/L, p < 0.001. The level of MR-proADM was gradually increased according to the dosage of furosemide as it is displayed in The level of the MR-proADM was also related to the dose of furosemide. We have discovered that patients taking higher doses of diuretics are having a statistically significant higher level of MR-proADM. The mean level of MR-proADM in patients with no furosemide  .In our study, we have also proved the prognostic value of MR-proADM in 24 months of follow-up and our cut-off value corresponds to the value of Haeling. Our results also reveal a strong correlation between the level of MR-proADM and the severity of the disease. Our previous studies have also shown that the predictive value of MR-proADM is strongest in patients with low comorbidities according to the AHEAD score. AHEAD is a simple scoring system based on the analysis of comorbidities; A= atrial fibrillation, H = hemoglobin, E = elderly, A = abnormal renal function, and D = diabetes mellitus. The predictive value of MR-proADM declined with a higher level of AHEAD ,13.Many studies have shown that the main reason for rehospitalization for heart failure is related to pulmonary congestion ,15,16. TADM could be one of these markers due to its pathophysiological properties. ADM levels rapidly decrease after treatment with diuretics. Persistent higher levels of ADM are associated with serious residual congestion and also a more severe prognosis. It could be suggested that patients who require higher doses of diuretics to manage their congestion are those in a more advanced condition of the disease, than those who require lower doses of loop diuretics based on their plasma level of MR-proADM. It can be concluded that higher levels of MR-proADM combined with increased use of loop diuretics are associated with a higher risk of serious progress of the disease. It may help to detect patients that are inadequately decongested or are resistant to diuretic therapy to re-evaluate their treatment strategy, such as changing to another loop diuretic, using a combination of diuretics, or using some of the elimination methods ,21. OtheElevated plasma levels of MR-proADM in patients with chronic HF reflect residual congestion and are associated with an increased risk of mortality and hospitalization.Our study has several limitations. The FAR NHL study population is a Czech multicentric cohort which might not resemble the heterogeneity of all patient populations. Only patients with mid-range and reduced ejection fractions were evaluated, and patients with heart failure with preserved ejection fractions were not included in the study. Basic laboratory measurements including NT-proBNP were not performed in a core laboratory and may be subject to between-laboratory variability. We measured the stable part of the ADM precursor peptide, mid-regional pro-ADM (MR-proADM), however, the level of biologically active amidated ADM (bio-ADM) might be more accurate in the assessment of the endogenous level of ADM .Adrenomedullin is a novel biomarker that is released as a counteracting response to volume overload. Mid-regional proADM is a stable and reliable surrogate for measurement markers of ADM release.MR-proADM also appears as a predictor of a more serious prognosis in two years follow-up. Higher levels of MR-proADM combined with increased use of loop diuretics are associated with a higher risk of serious progress of the disease and may refer to a reassessment of the treatment strategy.


Ka range of 2.6–23.51 shows a dependence upon the pKa of the acid when the acid pKa falls between 8 and 20 (in acetonitrile). A potential-pKa diagram is constructed and used to estimate the pKa of the azo-OMe species. Heterogeneous electron-transfer rate constants are obtained using rotating disk electrode voltammetry in combination with Koutecký–Levich and Tafel analysis. Electrochemical analysis shows that the reactions are diffusion limited and are kinetically controlled by the electron-transfer step. Kinetic isotope studies indicate a concerted proton, electron transfer event occurs in the pKa-dependent range when using trifluoroacetic acid.Herein, we report the thermodynamics, kinetics, and mechanism for electrochemical proton-coupled electron transfer involving the anthracene-based azo dye azo-OMe. The peak reduction potential of azo-OMe with organic acids spanning the p Herein, we report the thermodynamics, kinetics, and mechanism for electrochemical proton-coupled electron transfer involving the anthracene-based azo dye azo-OMe. Fundamental mechanistic studies, wherein the PCET mechanism can be controlled, are of vast importance for energy conversion reactions.Nature has evolved a mechanistic advantage in carrying out electron-transfer steps, as well as associated chemical reactions, by coupling the movement of electrons along reaction pathways to proton motions. The coupling of proton and electron movements play an important role in energy conversion reactions, energy storage, and small molecule activation reactions.6–15 Experimental, theoretical, and computational work has shown non-innocence of protons at electrochemical interfaces.14–25 Indeed, many chemical reactions, including reactions involving homogeneous catalysis and heterogeneous catalysis at electrode surfaces show a clear dependence on the pH of the solution.15Over the past decade, PCET at electrochemical interfaces has gained attention due to the significant role electrochemical PCET plays in energy conversion reactions and catalysis.5,26–29Protons influence the Fermi level at the electrochemical interface thereby controlling the thermodynamics and, as a result, the kinetics of electrochemical PCET reactions. Transition state theory makes this connection between activation energy and reaction kinetics. The activation energy of stepwise PCET compared to the activation energy of concerted PCET dictates the observed mechanism. For example, if the activation energy of the concerted PCET reaction is less than the activation energy of the sum of the individual proton- and electron-transfer steps, then the concerted reaction will be kinetically favoured. It follows, then that the dependence of the reactions on pH results from a decoupling of the electron and proton transfer steps in PCET reactions caused by one step of the reaction (ET or PT) becoming more thermodynamically favoured. With this in mind, catalysts and reactions can be designed such that changes in pH result in changing the mechanism of PCET between stepwise or concerted PCET.Ka of organic acids, the rate and mechanism of PCET in small-molecule inorganic complexes can be systematically dialled in.22,23,30,31 Further, strong electronic coupling between molecular sites on electrodes has been shown to cause reactivity that fundamentally diverges from that of solution-phase or surface-tethered analogues and can be correlated to the acid/base behaviour of the molecular analogue.17 Many recent mechanistic studies involved photo-induced PCET of bio-mimic model systems.32 Photo-induced PCET model systems, both molecular and biological, have been used to quantify the influence of proton-transfer driving force and transfer distance on the kinetics of PCET.33–38 Because of the complex nature of PCET, it remains important to understand mechanisms and factors that govern electron and proton transfer at electrochemical interfaces of a wide variety of molecular and materials systems.The nature of the proton  can be the driver for tuning the thermodynamics of the proton-transfer step in the chemical reactivity. By tuning the p39–42Synthetically facile molecular model systems are important to form the basis for the study for PCET. Azo dyes possess such facile synthetic procedures that offer rich synthetic versatility and allow for the development of a wide range of model systems in which thermodynamic properties can be systematically tuned. Applications of azo dyes include organic solar cells, molecular photoswitches, protein probes, molecular machines, and non-linear optical components, which encompasses a wide range of applications-driven systems.versus 0 eq. TFA.43 This work builds upon these initial electrochemical measurements to fully understand the electrochemical PCET reactivity of this anthracene-based azo dye. Towards this end, we have probed the electrochemical reaction of azo-OMe using several different organic acids with acidity constants spanning the pKa range of 2.6–23.51 in acetonitrile using the combination of cyclic voltammetry, differential pulse voltammetry, and rotating-disk electrode voltammetry along with a kinetic isotope dependence study. Each of the electrochemical reduction reactions are show to be diffusion controlled at the electrode surface, and are kinetically limited by the electron-transfer step. We show that the PCET mechanism is tuned by the variation of the organic acid pKa, which serves a proxy (or stand in) for pH in non-aqueous conditions. Using a combination of rotating disk voltammetry and kinetic isotope effect studies, we show that in solutions with a less acidic acid , azo-OMe undergoes a concerted 1H+/1e− transfer PCET redox reaction. The activation energy of the concerted reaction is less than the sum of the individual proton- and electron-transfer steps. However, in the presence of a highly acidic organic acid , such as HDMF OTf, a proton transfer occurs prior to the electrochemical electron transfer, indicating that the activation energy of the proton-transfer step is sufficiently decreased to decouple the concerted PCET reaction.In previous work from our group, we described the design and study of (anthracen-2-yl)-2-(4-methoxyphenyl)diazene azo-OMe, . as a moKa range in acetonitrile of 2.6–23.51  that yields a complete ET/PT thermodynamic picture of azo-OMe. A 10 mM stock solution of acid with 0.5 mM azo-OMe in acetonitrile (100 mM TBAPF6) was titrated into a solution of 0.5 mM azo-OMe (100 mM TBAPF6) at 100 and 500 μL increments (0.19–2.33 eq. of acid). CV was acquired upon each addition of acid and DPV was acquired at 1 and 2 eq. of acid.The redox properties of azo-OMe were measured with cyclic voltammetry (CV) and differential pulse voltammetry (DPV). To probe the electrochemical PCET reaction, nine organic acids were used, with a pE1/2 = −1.7 V vs. Fc+/0 before addition of any acid.43 Upon the addition of small amounts  of each acid, the reversible reduction peak disappeared concomitant with the growth of an irreversible reduction peak at more positive potentials. As additional acid was added , the initial reversible peak continued to disappear and the first irreversible reduction peak also disappeared to yield another, even more positively shifted irreversible reduction peak The irreversible peaks were shifted to positive potentials, meaning that reduction became more facile upon the addition of acid  resulted in a smaller positive shift of the reduction potential. For example, addition of acetic acid (pKa (MeCN) = 23.51) resulted in only a 300 mV shift from E1/2 = −1.7 V vs. Fc+/0 (no added acid) to E1/2 = −1.38 V vs. Fc+/0 upon addition of 2 equivalents of acid. Whereas, acids with lower pKa values (in acetonitrile) resulted in significantly larger changes in the reduction potential. Specifically, dimethylformamidium trifluoromethanesulfonic acid (HDMF) (pKa (MeCN) = 2.6) resulted in a 950 mV shift to E1/2 = −0.45 V vs. Fc+/0 upon addition of 2 equivalents of acid. By utilizing organic acids with pKa values spanning this range, the thermodynamics of azo-OMe and any reactions that occur using it can be experimentally determined.As expected, the ΔKa diagrams have been developed to understand PCET reactions in non-aqueous solutions for which Pourbaix diagrams that are used for aqueous systems cannot be employed. Because solvated protons do not exist in organic solvents the way they do in aqueous media, the pH scale of a Pourbaix diagram must be modified. Instead of pH, the pKa of an organic acid can be used as the proxy for the thermodynamic availability of the proton in a potential-pKa diagram.44 The potential-pKa diagram plots the electrochemical reduction or oxidation potential of a process of interest versus the pKa of a range of added organic acids. These plots show regions in which various electrochemical and protonation states of the analyte, in this case the azo-OMe, exist as a function of proton availability (from an added organic acid).Potential-pKa diagram for azo-OMe, the reduction potential of azo-OMe was measured using cyclic voltammograms that were acquired with 0.5 mM azo-OMe, 2 equivalents of acid, and 100 mM TBAPF6 in acetonitrile. vs. Fc+/0) plotted versus the corresponding pKa of each added acid. The potential-pKa diagram for azo-OMe reveals one pKa-dependent and two pKa-independent regions. One pKa-independent region occurred when strongly acidic acids (pKa 2.6 to 8.6) are used. In this region, the results suggests that azo-OMe is protonated prior to reduction and that in this case, the reduction potential of the protonated azo-OMe dye is measured directly. The other pKa-independent region occurred when weaker acids (pKa 20.35 to 23.51) were used. In this region azo-OMe is likely not significantly protonated prior to or during the reduction event. The acids with pKa > 20.35 are not acidic enough to protonate either the reduced or neutral azo-OMe species. A pKa-dependent region is observed between (pKa 2.6 to 8.6). The slope of the pKa-dependent region (pKa 8.6 to 20.35) is 75 mV per decade, which indicates that this process involves 1H+/1e− transfer event. The difference from the idealized Nernstian slope (59 mV per decade) is not substantially large and due to homoconjugation of organic acids in acetonitrile.44 While the process could be a 2H+/2e− process, the current response associated with the azo-OMe PCET reaction does not in fact double when the acid concentration is increased from 1 to 2 equivalents of acid, thereby ruling out that possibility.To construct the potential-pKa dependent and independent regions can be used to predict the pKa of [azo-OMe(H)]+ and [azo-OMe(H)], which are approximately 8.6 and 20.1 respectively. Taken together, lines drawn through each pKa dependent . HDMF OTf and TFA were chosen because they fall into the two relevant pKa ranges for PCET reactions with azo-OMe, according to the potential-pKa plot. HDMF OTf is highly acidic and protonates azo-OMe even when not reduced, and TFA falls into the pKa-dependent region and is acidic enough to protonate the reduced azo-OMe species. vs. Fc+/0. For the 2 eq. TFA voltammograms, the onset of current is shifted to more reducing potentials (∼−0.6 V vs. Fc+/0 rather than −0.2 V vs. Fc+/0 for the HDMF voltammograms). The plateau current was not observed in the RDE plot with 2 eq. of TFA because proton reduction occurs via the glassy carbon electrode at −1.2 V vs. Fc+/0, which begins to dominate the current response. The disk current of azo-OMe with 2 eq. of HDMF OTf and 2 eq. of TFA exhibited Levich behaviour, which is the increase in current response as the rotation rate is increased. The Levich behaviour observed when the rotation rate was increased (vide infra).To garner mechanistic understanding of electrochemical PCET processes, rotating disk electrode (RDE) voltammograms were acquired with solutions of 0.5 mM azo-OMe and 2 eq. of acid  at various rotation rates , B is the Levich constant, ω is the angular rotation rate (rad s−1), and iK is the kinetic current. The KL plots of azo-OMe with 2 eq. of HDMF OTf and azo-OMe with 2 eq. TFA are shown in The data obtained from RDE experiments were plotted using the Koutecký–Levich (KL) eqn (1)versus the reciprocal of the square root of rotation rate. The reaction overpotential (η) is defined as the difference between the peak potential of azo-OMe0/− (E) in the presence of acid and the half potential of azo-OMe0/− (−1.7 V = Eeq.) The reciprocal of measured currents, obtained from the rotating-disk voltammograms over a range of overpotentials that fall within the plateau region, were plotted y-axis, which indicates that the reaction is also kinetically limited by the electron-transfer step. The linear KL plots were extrapolated to yield the y-intercept, 1/iK, where current would be kinetically limited, but not limited under diffusion control. The separation of the kinetically-limited and diffusion-limited processes using KL treatment allows for the determination of heterogeneous electron-transfer rate constants.The KL plots at various overpotentials do not intersect the origin of the i0, log iK (iK = inverse of the KL y-intercept) was plotted versus overpotential, shown in η is overpotential, A is the Tafel slope, iK is the kinetic current, and i0 is the exchange current. We can use the exchange current to calculate the heterogeneous electron-transfer rate constant, ks, which was obtained using F is Faraday constant, A is surface area of the electrode, C is the concentration of azo-OMe, and ks is the heterogeneous electron-transfer rate constant. The heterogeneous rate constants obtained are summarized in To obtain the exchange current, ks = 0.614 ± 0.008 cm s−1) than the electron transfer than when 2 equivalents of TFA with azo-OMe is used (ks = 0.587 ± 0.005 cm s−1). This observation can be understood by considering the thermodynamics of proton transfer before the electron transfer is initiated. The pKa (MeCN) of HDMF OTf is 2.6, four orders of magnitude more acidic than the azo-OMe, which is estimated to be 8.6 from the potential-pKa analysis (Ka of TFA (pKa = 12.6 in MeCN) is less than that of azo-OMe, meaning that the azo-OMe is not appreciably protonated before the electron transfer event. However, upon reduction, the azo-OMe will be certainly be protonated (pKa [azo-OMe]− = ∼20 in MeCN) because the reduced species is far less acidic than the TFA. Because both the electron and proton transfer must occur in this event, the electron transfer is ks = 0.587 ± 0.005 cm s−1, slower than the more acidic solutions. With 2 equivalents of TFA, azo-OMe is not protonated prior to electron transfer, but from this analysis, it is not clear whether there is concerted electron–proton transfer (CPET) or stepwise electron, then proton transfer (ETPT). To distinguish between these processes, kinetic isotope experiments were performed.In solutions of 2 equivalents HDMF OTf with azo-OMe, the electron transfer is found to be faster  of 1.8 was found, which is consistent with a concerted proton- and electron-transfer mechanism.24,32 Previous reports by Costentin et al. indicated an intramolecular electrochemical CPET of a substituted-phenol with a nearby amine group (KIE of 1.8).45A KIE ]+ (≈8.6 in MeCN) and weak organic acids, such as TFA (ΔpKa = pKa(TFA) − pKa([azo-OMe(H)]+) ≈ 4). A protonation prior to an electron-transfer in this regime would be too energetically uphill and a concerted mechanism would bypass that high energy intermediate. An ETPT mechanism can be excluded based on the magnitude of KIE , which is consistent with the involvement of PT in the rate-determining electron transfer step.46,47Along the p43 In this work, we have probed the proton-dependence of the electrochemical reaction of azo-OMe using nine organic acids, with acidity constants spanning the pKa range of 2.6–23.51 in acetonitrile, using a combination of electrochemical techniques. Our goal was to control the kinetics and mechanism of the PCET reaction at an electrode surface by tuning the thermodynamic (and by extension of transition state theory) the kinetics of the PCET reaction.To understand the electrochemical PCET reactivity, this work builds upon initial electrochemical measurements of azo-OMe previously reported by our laboratory.Ka diagram, which indicated a 1H+/1e− PCET process occurrs in the presence of acids with pKa values spanning pKa 8 to 20. The potential-pKa diagram was also used to predict the pKa of [azo-OMe(H)]+ and [azo-OMe(H)], which are approximately 8.6 and 20.1 respectively.The data obtain from cyclic voltammetry experiments was used to construct a potential-pKa difference between azo-OMe and HDMF OTf (pKa (MeCN) = 2.6), azo-OMe is overwhelmingly protonated in solution of 2 equivalents HDMF OTf prior to an electron transfer event, which allows for a faster electron transfer (ks = 0.614 ± 0.008 cm s−1). In solution of 2 equivalents TFA (pKa (MeCN) = 12.6), ks = 0.587 ± 0.005 cm s−1, slower than the more acidic solutions. To understand this result, kinetic isotope effect studies were conducted with deuterated TFA to distinguish if the PCET event in the less acidic TFA was concerted (CPET) or stepwise (ETPT). A KIE of 1.8 was obtained, which indicates that the electron and proton are transferred in a concerted step.Rotating disk voltammetry with Koutecký–Levich and Tafel analysis was used to obtain a mechanistic understanding of the PCET process azo-OMe undergoes in the presence of added organic acids. These analyses shows that the PCET reactions are diffusion limited and are kinetically controlled by the electron-transfer step. Due to the pvia an intramolecular processes, which have been studied in a limited fashion prior to this.20 Ongoing efforts to vary the identity of arene on the anthracene side of the azo bond as well as the R group on the phenyl side are currently underway. Further, work is underway to probe the influence of azo bond protonation on the ultra-fast excited state dynamics of the azo dyes for which the thermodynamic properties reported in this work are essential.Our results demonstrate that azo-based dyes exhibit rich electrochemical PCET chemistry – the mechanism of which can be tuned using the proton-transfer thermodynamics. These results represent some of the first examples using azo-based dyes that examine the PCET mechanism at electrode surfaces in which the proton originates from organic acids introduced into the solution, rather than 43All reagents were obtained from commercial sources and used as received without further purification, unless otherwise specified. The anthracene-based azo dye, (anthracen-2-yl)-2-(4-methoxyphenyl)diazene (azo-OMe) was synthesized according to literature procedure.6) was recrystallized with hot ethanol three times, dried under vacuum, and used as the supporting electrolyte. Acetonitrile was pre-dried with 4 Å molecular sieves, distilled over calcium hydride, and stored over 4 Å molecular sieves prior to use. Ferrocene was used as an internal standard and all voltammograms were reported vs. Fc+/0 couple. The scan rate used for CV was 100 mV s−1 unless otherwise specified. All samples were sparged with N2 for 10 minutes before CV and DPV and kept under a N2 blanket during acquisition.All cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements were recorded on a CH Instruments potentiostat/galvanostat. CV and DPV were performed using a glassy carbon disk (3 mm diameter) working electrode, a platinum wire counter electrode, and a silver wire quasi reference electrode. The glassy carbon working electrode was polished with 0.05 μM alumina powder/water slurry, rinsed with water and acetone, and allowed to air dry prior to each experiment. Tetrabutylammonium hexafluorophospate (TBAPF−1 with rotation rates (ω) of 100, 200, 500, 1000, 1500, and 2000 rpm. All solutions were sparged with N2 for 10 minutes before RDE and were blanketed with N2 during acquisition.All rotating disk experiments were acquired with a Bio-Logic SP-300 bipotentiostat/galvanostat and a Pine MSR rotator. A glassy carbon fixed-disk PEEK shroud working electrode and a platinum mesh counter electrode were used for RDE experiments. The glassy carbon disk was polished with 0.05 μM alumina powder/water slurry, rinsed with water and acetone, and allowed to air dry prior to each experiment. Acetonitrile was purified from a Pure Process Technology free standing solvent purification system. The scan rate used for RDE was 20 mV sThere are no conflicts to declare.RA-010-D0RA01643H-s001


WHO estimates that seasonal influenza epidemics result in three to five million cases of severe illness  every year. We aimed to improve the understanding of influenza-associated hospitalisation estimates at a national and global level.We performed a systematic literature review of English- and Chinese-language studies published between 1995 and 2020 estimating influenza-associated hospitalisation. We included a total of 127 studies (seven in Chinese) in the meta-analysis and analyzed their data using a logit-logistic regression model to understand the influence of five study factors and produce national and global estimates by age groups. The five study factors assessed were: 1) the method used to calculate the influenza-associated hospitalisation estimates (rate- or time series regression-based), 2) the outcome measure , 3) whether every case was laboratory-confirmed or not, 4) whether the estimates were national or sub-national, 5) whether the rates were based on a single year or multiple years.The overall pooled influenza-associated hospitalisation rate was 40.5  = 24.3-67.4) per 100 000 persons, with rates varying substantially by age: 224.0 (95% CI = 118.8-420.0) in children aged 0-4 years and 96.8 (95% CI = 57.0-164.3) in the elderly aged >65 years. The overall pooled hospitalisation rates varied by calculation method; for all ages, the rates were significantly higher when they were based on rate-based methods or calculated on a single season and significantly lower when cases were laboratory-confirmed. The national hospitalisation rates  varied considerably, ranging from 11.7 (95% CI = 3.8-36.3) per 100 000 in New Zealand to 122.1 (95% CI = 41.5-358.4) per 100 000 in India .Using the pooled global influenza-associated hospitalisation rate, we estimate that seasonal influenza epidemics result in 3.2 million cases of severe illness  per annum. More extensive analyses are required to assess the influence of other factors on the estimates (e.g. vaccination and dominant virus (sub)types) and efforts to harmonize the methods should be encouraged. Our study highlights the high rates of influenza-associated hospitalisations in children aged 0-4 years and the elderly aged 65+ years. Influenza is a common respiratory disease with high incidence rates reported in countries worldwide ,2. DespiWHO estimates that annual influenza epidemics result in roughly three to five million cases of severe illness  annuallA recent literature review of influenza-associated hospitalisation rates (from 2007 to 2018) identified variability in the methods, case definitions, and data sources, concluding that calculating a pooled estimate was impossible due to extremely high heterogeneity in estimates observed across studies . While wWe searched MEDLINE, Embase, CNKI, Wanfang, and Chongqing VIP (with the last three being Chinese databases) for English- and Chinese-language studies, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The search included a combination of search terms related to 1) hospitalisation, 2) influenza and 3) “influenza associated” or “excess”. Details of the literature search can be found in the We included studies that were published between 1995 and the end of March 2020, that reported hospitalisation rates for the general population, and that focused on total influenza (2009 pandemic estimates were excluded). We pooled estimates (where possible) with an influenza (sub)type focus. Studies from all countries and/or regions were eligible and there was no restriction by age groups (for the literature review). Two types of studies were eligible for inclusion in the literature review: rate-based studies and time series regression-based studies. Rate-based studies were defined as studies that report age group-specific hospitalisation rates for influenza using primary data  and applying these to a population denominator, ie, catchment population. Time series regression model studies were defined as studies that estimate excess hospitalisations (using regression methods). A full and detailed description of the literature review can be found in the When extracting the data, we characterized the studies based on five factors that are readily available in all papers: 1) method (rate- or time series regression-based), 2) outcome measure , 3) laboratory-confirmed or not, 4) national or sub-national data, and 5) if the rates were based on a single year or multiple years. The “narrow” envelope was based on laboratory confirmation and a clinical diagnosis of influenza, the “medium” envelope was based on broad definitions of respiratory syndromes  without laboratory confirmation, and the “wide” envelope was defined as broader disease categories that can be associated with influenza , without laboratory confirmation  logit-logistic regression model to generate national and global estimates of influenza-associated hospitalisations ,12. The We estimated all models using restricted iterative generalized least squares (RIGLS) with first-order penalised quasi-likelihood (PQL). The overall pooled hospitalisation rate was equally weighted for the five factors and for categories within a factor. No weight was added for differences in sample size, as is very often done in meta-analyses, primarily because the heterogeneity between individual measures and studies was so large that weighting for sample size would make the different model estimates significantly more difficult to interpret.We calculated country estimates using an empirical-Bayes procedure in the multilevel models. Notably, a country estimate can differ strongly from the published country rate as the country estimates are adjusted for the five factors included in the model, like the overall pooled estimated rates. For every age category  a separate model was estimated.The English literature search retrieved a total of 4625 records. After deduplication, 3906 records remained for title and abstract screening, after which 312 records remained for full-text review. A total of 120 studies were included for data extraction. The Chinese literature search retrieved 3046 records. After deduplication, 2657 records were screened by title and abstract, 24 of which proceeded to the full-text screening. A total of seven studies were included for data extraction , with most estimates being rejected due to not fitting the selected age groups or having no information available on 95% confidence intervals (CIs) or standard errors (SEs). The 96 estimates came from a total of 40 countries/administrative regions   per 100 000 persons, with rates varying substantially by age: 224.0 (95% CI = 118.8-420.0) in children aged 0-4 and 96.8 (95% CI = 57.0-164.3) in the elderly aged >65 , laboratory-confirmed studies tended to have lower rates (vs non-laboratory confirmed studies) and a broader outcome measure had higher rates. An important advantage of applying our modeling approach is that the country and global estimates are more comparable, as the method assumes they were calculated with the same (five) study characteristics. Using this approach, the national hospitalisation rates varied widely, ranging from 11.7 (95% CI = 3.8-36.3) per 100 000 in New Zealand to 122.1 (95% CI = 41.5-358.4) per 100 000 in India . The global pooled influenza-associated hospitalisation rate using all studies was 40.5 (95% CI = 24.3-67.4) per 100 000 population, with rates varying substantially by age: 224.0 (95% CI = 118.8-420.0) per 100 000 in children (aged 0-4) and 96.8 (95% CI = 57.0-164.3) in the elderly (>65).Our study used a similar design to a previous study which investigated influenza-associated hospitalisation rates worldwide . HoweverNotably, our choice of the five factors was based on what could be directly extracted from the 127 papers identified in our literature review, which was reflected in the high levels of unexplained heterogeneity in our models. In the future, other factors should be considered for this type of analysis, including: vaccination coverage rates, co-morbidities, the underlying population distributions (e.g. percentage of the population aged under 15 years ), the ciThis study can be seen as a pilot study, as it only focuses on the influence of five factors on influenza-associated hospitalisation rates. A larger study should be performed which extends the analysis to a wider range of factors , as this would allow for a better definition and understanding of the heterogeneity of the rates and for improving the pooled estimates. Ideally, researchers would use this approach to make national, regional, and global estimates, requiring sufficient national estimates from each world region. Importantly, our study highlights the need for high quality, and harmonized  estimates of influenza-associated hospitalisation rates, as these are important for research and policy means.We gathered the influenza-associated hospitalisation rates based on two general approaches: rate-based estimates and time series regression-based estimates. These two methods are inherently different, as the first is typically based on hospital data (with cases often laboratory-confirmed) and the second is a modeling exercise (using various linear regression methods ) where eA striking finding of our analysis is that, while the US had an influenza-associated hospitalisation age signature that had the highest rates in the elderly (>65), the highest rates in all other countries, after adjusting for the five factors, were in children aged 0-4 years, in both high- and low-income countries (Another Burden of Influenza and RSV Disease (BIRD) study modeled the impact of influenza and respiratory syntactical virus (RSV) on hospitalisation admission rates based on ICD codes for acute respiratory infections and the proportion of cases that were RSV- and influenza-positive (laboratory-confirmed) . By modeOur pooled estimates of influenza-associated hospitalisations can be used as a reference for comparison purposes, either at a national  or aOur study has several strengths and limitations. Two important strengths are that we also reviewed the Chinese-language literature (3046 papers based on our inclusion criteria) and our modeling approach allows one to assess the influence of five factors on the hospitalisation estimates and to calculate pooled estimates. Our literature review was also more extensive than the previous one ). One liOur study highlights systematic variation in influenza-associated hospitalisation estimates around the world and the importance of assessing the hospitalisation rates by age group. Because the estimation of influenza-associated hospitalisation rates is not straightforward and entails assumptions that are difficult to test, it is important to compare estimates from different modeling approaches, as we have done here. This information can be used to improve estimates of the burden of severe diseases, especially in lower- and middle-income countries, given their importance for vaccination program policies. Importantly, our estimate of 3.2 million hospitalisations fits the WHO estimates that annual influenza epidemics result in three to five million annual cases of severe illness  per annum ,5. Final